Toxicology studies of substances have traditionally relied on unicellular (for example, the Ames test or the yeast carcinogenic assay described in U.S. Pat. No. 4,997,757) or in vitro systems for toxicity testing and the prediction of human risk. However, there are many factors that make it difficult to extrapolate from such data to human risk including cellular affinity of the substance, uptake and distribution differences between single cells and whole animals, metabolism of the substance, and cascade effects where the effect of the substance is mediated through a cellular process. These same factors can affect the progress of pharmaceutical research and development as well when attempting to determining and/or predicting the effects of a compound in an animal system.
Further, the end-point of traditional animal based toxicology studies is typically determination of an LD50 (the dose at which 50% of the test animals die). Dead animals may be subjected to further analysis, for example, histopathology, but such analysis is generally labor intensive and relatively insensitive.
MacGregor, et al (Fundamental and Applied Toxicology, 26:156-173, 1995) have reviewed molecular end-points and methods of routine toxicity testing including the following: damage-inducible genes in individual cells; bacterial models of toxicity; screening of stress-gene expression using hybridization or polymerase chain reaction; hybridization probes for detection of chromosomal aberrations; single cell electrophoresis assays; and in vivo animal studies involving animal sacrifice and subsequent analysis of tissue/cellular damage.
General strategies for generating transgenic (Tg) animals has been well described (Pinkert, C. A. (ed.) 1994. Transgenic animal technology: A laboratory handbook. Academic Press, Inc., San Diedo, Calif.; Monastersky G. M. andRobl, J. M. (ed.) (1995) Strategies in transgenic animal science. ASM Press. Washington D.C.), however, the time-consuming processes of screening for presence and function of the transgene remained rate limiting steps. These screens typically involve conventional assays, PCR, Southern blot hybridization or slot-blot hybridization (Tinkle, B. T. et al., (1994) In Pinkert, C. A. ed., Transgenic animal technology: A laboratory handbook, pp. 221-34 Academic Press, Inc., San Diego, Calif.), to demonstrate the presence of integrated DNA. To determine if the gene products are expressed steady-state levels of mRNA transcripts can be assessed by Northern blot hybridization, RT-PCR, or in situ hybridization, and protein expression assayed using Western blots, or immunofluorescent staining. Finally, a wide range of assays are needed to determine function as an indication of the appropriate phenotype. Tissues from the Tg animal are required to perform these molecular and functional analyses (Bieberich, C. J., et al., (1994) In Pinkert, C. A. ed., Transgenic animal technology: A laboratory handbook, pp. 235-62. Academic Press, Inc., San Diego, Calif.), and removal of tissues may not be possible until a line is established. Thus, a rapid noninvasive screening method is needed as a functional assay for the generation and study of Tg animals.
A wide range of Tg mice that employ reporter constructs have been developed and tested. For example, Tg mice containing viral long terminal repeat (LTR) promoter fusions have been used to study the range of tissues and cell types that are capable of supporting HTLV-1 expression and the development of neurofibromatosis-like tumors associated with HTLV-1 retrovirus (Bieberich, C. J., et al., (1993) Virol. 196:309-18.). The LTR from HIV-1 has been fused to luciferase to evaluate transcriptional regulation by UV light and various sensitizing agents (Morrey, J. D., et al., (1992) J. Acquir. Immune Defic. Syndr. 5: 1195-203; Morrey, J. D., et al., (1991) J Viol. 65: 5045-51). Cardiovascular biology and diseases have been investigated in Tg mouse models using tissue-specific promoters (Johnson, J., et al., (1989) Mol. Cell. Biol. 9: 3393-9; Rindt, H., et al., (1993) J. Biol. Chem. 268: 5332-8; Seidman, C. E., et al., (1991) Can. J. Physiol.Pharmacol. 69: 1486-92; Tsika, R. W., et al., (1990) Proc. Natl. Acad. Sci. USA. 87: 379-83), and regulation of insulin-responsive glucose transporter GLUT4 and Apo A-I genes have also studied in models of diabetes, obesity (Liu, M. L., et al., (1992) J. Biol. Chem. 267: 11673-6) and coronary artery disease (Walsh, A., et al., (1993) J. Lipid Res. 34: 617-23; Walsh, A., et al., (1989) J. Biol. Chem. 264: 6488-94).
Photoproteins as biological labels have been used for more than a decade for the study of gene expression in cell culture or using excised tissues (Campbell, A. K. 1988. Chemiluminescence. Principles and applications in biology and medicine. Ellis Horwood Ltd. and VCH Verlagsgesellschaft mbH, Chichester, England; Hastings, J. W. (1996) Gene. 173:5-11; Morrey, J. D., et al., (1992) J. Acquir. Immune Defic. Syndr. 5: 1195-203; Morrey, J. D., et al., (1991) J Viol. 65: 5045-51.). Low-light imaging of internal bioluminescent signals has been used to study temporal and spatial gene regulation in relatively thin or nearly transparent organisms (Millar A. J., et al., (1992) Plant Cell 4:1075-87; Stanewsky, R., et al., (1997) EMBO J. 16:5006-18; Brandes C, et al., (1996) Neuron 16:687-92). External detection of internal light penetrating the opaque animal tissues has been described (Contag, P. R., et al., (1998) Nature Med. 4:245-7; Contag, C. H., et al., (1997) Photochem Photobiol. 66:523-31; Contag, C. H., et al., (1995) Mol Microbiol. 18:593-603).
However, heretofore no in vivo screening method has been described that allows screening of compounds in whole, live animals where real-time data could be collected concerning the effects of a test substance on, for example, specific aspects of toxicity and substance metabolism.